Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure
Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure
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DOI：
10.1016/j.exphem.2009.07.012
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发表时间：
2009-11-01
影响因子：
2.6
通讯作者：
Yao, Chao-Ling
中科院分区：
文献类型：
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作者：
Chen, Te-Wei;Hwang, Shiaw-Min;Yao, Chao-Ling
Objective.在无血清条件下从造血干细胞大量产生巨核细胞的完整过程对于血小板减少症患者的快速血小板重建具有巨大的临床潜力。我们以前曾报道过一种优化的无血清培养基（无血清造血干细胞培养基）的产生，用于CD 34（+）细胞的体外扩增。在这里，我们进一步从无血清扩增的CD 34（+）细胞在完全和最佳的无血清条件下产生大量的功能性巨核细胞，以符合临床规定。采用系统的方法筛选并优化巨核细胞生成所需的血清代用品和细胞因子的浓度。通过检测非肥胖糖尿病/严重联合免疫缺陷小鼠巨核细胞的表面抗原、基因表达、体外巨核细胞活化能力、巨核细胞和血小板的恢复能力等指标，对无血清诱导的巨核细胞进行鉴定。最佳的无血清巨核细胞诱导培养基是含有血清替代物的Iscove改良Dulbecco培养基（即，人血清白蛋白、人胰岛素和人转铁蛋白）和细胞因子混合物（即，血小板生成素、干细胞因子、Fms样酪氨酸激酶3配体、白细胞介素-3、白细胞介素-6、白细胞介素-9和粒细胞-巨噬细胞集落刺激因子）。诱导后的巨核细胞表达CD 41 a和CD 61表面抗原、核因子红系衍生2和加塔-1转录因子以及巨核细胞活化能力。结论：诱导巨核细胞移植可促进受照非肥胖糖尿病/严重联合免疫缺陷小鼠巨核细胞和血小板的恢复。综上所述，我们开发了一种无血清巨核细胞诱导培养基，无血清巨核细胞和无血清造血干细胞培养基的组合可以高效地产生大量功能性巨核细胞。我们的方法代表了巨核细胞和血小板的未来细胞治疗的一个有前途的来源。(C)2009 ISEH -血液学和干细胞学会。爱思唯尔公司出版
Objective. A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations.Materials and Methods. Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in non-obese diabetic/severe combined immunodeficient mice.Results. The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interieukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated non-obese diabetic/severe combined immunodeficient mice.Conclusion. In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.